JP2017049024A - Preparation method of sample for advanced glycation end product analysis, and analytical method of advanced glycation end product - Google Patents
Preparation method of sample for advanced glycation end product analysis, and analytical method of advanced glycation end product Download PDFInfo
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Abstract
Description
本発明は、最終糖化産物の分析のための試料の前処理方法に関する。詳細には、生体試料中に遊離した最終糖化産物を極めて高感度・高精度で分析するための試料の前処理方法に関する。 The present invention relates to a sample pretreatment method for analysis of final glycation products. Specifically, the present invention relates to a sample pretreatment method for analyzing a final glycation product released in a biological sample with extremely high sensitivity and high accuracy.
従来、分子量１０００以下の生体由来の低分子を測定及び分析する場合、高速液体クロマトグラフィー（ＨＰＬＣ）と質量分析（ＭＳ）とを組み合わせたＬＣ−ＭＳやＬＣ−ＭＳ／ＭＳシステムが用いられている。また従来、生体試料をＬＣ−ＭＳ分析する場合は、一般に、生体試料をＨＰＬＣにアプライする前に、酸で加水分解した後、夾雑成分の除去及び分離のためのＣ１８担体等を用いた逆相カラム処理にかけることにより、分析対象物質の初発純度を向上させていた。しかし、対象物質によっては、前記逆相カラム処理では夾雑成分を十分に除去できないことがあり、そのため分析結果に多くのノイズが検出されて、十分な検出感度や精度が得られないことがあった。 Conventionally, when measuring and analyzing a biologically-derived small molecule having a molecular weight of 1000 or less, an LC-MS or LC-MS / MS system combining high performance liquid chromatography (HPLC) and mass spectrometry (MS) has been used. . Conventionally, when LC-MS analysis is performed on a biological sample, generally, the biological sample is hydrolyzed with an acid before being applied to HPLC, and then reversed-phase using a C18 carrier or the like for removing and separating contaminant components. The initial purity of the analyte was improved by subjecting it to column treatment. However, depending on the target substance, the reverse phase column treatment may not sufficiently remove contaminant components, so that a lot of noise is detected in the analysis result, and sufficient detection sensitivity and accuracy may not be obtained. .
より精度の高い分析を行うため、試料の前処理方法の改良が求められている。例えば、特許文献１には、生体試料中のデスモシンとイソデスモシンのＬＣ−ＭＳ／ＭＳ分析の測定精度を向上させるために、生体試料を、陽イオン交換樹脂にかけ、非酸性条件下で自然落下にて溶出させ、得られたサンプルをＬＣ−ＭＳ／ＭＳ分析にかける方法が記載されている。しかしながら、有効な前処理方法は物質によって異なることがあるため、測定する目的物質にとって適切な前処理方法を見出すことは容易ではない。
In order to perform analysis with higher accuracy, improvement of a sample pretreatment method is demanded. For example, in
ところで、最終糖化産物（Advanced Glycation Endproduct：ＡＧＥ）は、体内で蛋白質と糖との反応により生成される物質の総称であり、糖尿病合併症等の指標として知られている。従来、ＡＧＥの分析方法としては、分析対象のＡＧＥを認識・結合するモノクローナル抗体が利用できる場合は、それを利用したＥＬＩＳＡ測定法を用いることが可能であるが、そのようなモノクローナル抗体が確立できていない場合は、糖化された蛋白質を酸又はアルカリによって加水分解し、ＨＰＬＣ分析、ＬＣ−ＭＳ分析、ＬＣ−ＭＳ／ＭＳ分析などによりＡＧＥを検出し、標品と比較することで特定、定量する方法が行われている。ＡＧＥを精度良く検出及び定量できれば、糖尿病合併症等の疾患の診断や研究のために有利であるが、従来、正確な分析は容易ではなかった。 By the way, the final glycation end product (AGE) is a general term for substances produced by the reaction of protein and sugar in the body, and is known as an index of diabetic complications. Conventionally, as a method for analyzing AGE, when a monoclonal antibody that recognizes and binds to the AGE to be analyzed can be used, an ELISA measurement method using it can be used. However, such a monoclonal antibody can be established. If not, hydrolyze the glycated protein with acid or alkali, detect AGE by HPLC analysis, LC-MS analysis, LC-MS / MS analysis, etc., and identify and quantify by comparing with the standard The way is done. Accurate detection and quantification of AGE is advantageous for diagnosis and research of diseases such as diabetic complications, but accurate analysis has not been easy.
本発明者らは先に、高感度且つ高精度なＡＧＥの分析を可能にする試料の調製方法として、生体試料を液相で酸処理するステップ、及び、酸処理した試料を強酸性陽イオン交換樹脂に添加して非酸性条件下で溶出するステップを含む調製方法を提案した（特許文献２）。この調製方法によって得られた試料はＡＧＥの純度が高く、該試料をＬＣ−ＭＳ／ＭＳ分析にかけると、ノイズが大幅に低減された測定データが得られる。 The inventors of the present invention have previously described a step of acid-treating a biological sample in a liquid phase as a sample preparation method enabling highly sensitive and highly accurate analysis of AGE, and a strong acid cation exchange of the acid-treated sample. A preparation method including a step of adding to a resin and eluting under non-acidic conditions has been proposed (Patent Document 2). The sample obtained by this preparation method has high AGE purity, and when the sample is subjected to LC-MS / MS analysis, measurement data with greatly reduced noise can be obtained.
特許文献２に記載の試料の調製方法は、高感度且つ高精度なＡＧＥの分析を可能にする方法ではあるが、生体試料の酸処理を含むため、例えばアルギニン由来ＡＧＥの如き、加水分解に不安定なＡＧＥの分析には利用し難く、分析可能なＡＧＥの種類に制限がある。また、この調製方法は、生体試料中に遊離した最終糖化産物のみを測定するのは難しいという問題がある。加水分解に不安定なＡＧＥを含め、遊離体のＡＧＥを網羅的に分析し得る方法が要望されている。
The sample preparation method described in
また従来、ＮＨ、ＯＨ、ＣＯ等の極性官能基を有する物質の質量分析には、液体クロマトグラフから溶離する液体試料をエレクトロスプレープローブ（ＥＳＩプローブ）によりイオン化して質量分析計に導入する、エレクトロスプレーイオン化質量分析法が利用されており、ＡＧＥを含む試料の質量分析にもこの分析法が利用されているところ、ＡＧＥを含む液体試料のエレクトロスプレーイオン化質量分析法による分析中に、該試料に含まれていたアマドリ転位生成物が、ＥＳＩプローブにおいて該試料に加えられる熱によってＡＧＥへと変化してしまい、その結果、該試料に本来含まれていたＡＧＥについての分析の精度が低下するという問題があった。このような、質量分析法に起因するＡＧＥの分析精度の低下を解決し得る方法は未だ提供されていない。 Conventionally, for mass analysis of substances having polar functional groups such as NH, OH, and CO, a liquid sample eluted from a liquid chromatograph is ionized by an electrospray probe (ESI probe) and introduced into a mass spectrometer. Spray ionization mass spectrometry is used, and this analysis method is also used for mass analysis of a sample containing AGE. During analysis of a liquid sample containing AGE by electrospray ionization mass spectrometry, The Amadori rearrangement product contained is changed to AGE by the heat applied to the sample in the ESI probe, and as a result, the accuracy of analysis of the AGE originally contained in the sample is lowered. was there. There has not yet been provided a method that can solve such a decrease in analysis accuracy of AGE caused by mass spectrometry.
従って本発明は、遊離体の生体由来最終糖化産物（ＡＧＥ）の分析を高感度且つ高精度で網羅的に実施し得る、試料の調製方法を提供することに関する。 Therefore, the present invention relates to providing a sample preparation method that can comprehensively and highly sensitively and accurately carry out the analysis of the glycated end product derived from living organisms (AGE).
本発明者らは、ＥＳＩ質量分析計によるＡＧＥ分析のためのより高純度な試料を調製する方法について検討した結果、生体試料に対し、限外濾過膜による濾過処理によって膜非透過画分である高分子量の成分を除去する処理を行い、且つ還元処理を行い、さらに強酸性陽イオン交換樹脂を用いて精製し、得られた試料をＬＣ−ＭＳ／ＭＳ分析にかけることにより、測定データのノイズが大幅に低減され、高感度及び高精度で遊離体のＡＧＥを分析し得ることを知見した。 As a result of studying a method for preparing a sample with higher purity for AGE analysis using an ESI mass spectrometer, the present inventors have found that a biological sample is a membrane-impermeable fraction by filtration using an ultrafiltration membrane. Performing treatment to remove high molecular weight components, reducing treatment, purification using strong acid cation exchange resin, and subjecting the obtained sample to LC-MS / MS analysis, noise of measurement data Was significantly reduced, and it was found that free AGEs can be analyzed with high sensitivity and high accuracy.
本発明は、前記知見に基づきなされたもので、最終糖化産物分析のための試料の調製方法であって、生体試料に対し、限外濾過膜で濾過処理する工程、還元処理を施す工程、を含む試料の調製方法である。 The present invention has been made on the basis of the above knowledge, and is a method for preparing a sample for final glycation product analysis, comprising the steps of filtering a biological sample with an ultrafiltration membrane, and performing a reduction treatment. It is the preparation method of the sample which contains.
また本発明は、前記知見に基づきなされたもので、前記の本発明の調製方法により調製された試料を液体クロマトグラフィー−質量分析法によって分析する、最終糖化産物の分析方法である。 The present invention has been made based on the above findings, and is a method for analyzing a final saccharification product, in which a sample prepared by the preparation method of the present invention is analyzed by liquid chromatography-mass spectrometry.
本発明の試料の調製方法によれば、遊離体の生体由来最終糖化産物（ＡＧＥ）の分析を高感度且つ高精度で網羅的に実施し得る、試料の調製方法が提供される。
また、本発明の最終糖化産物の分析方法によれば、本発明の試料の調製方法によって調製された試料を液体クロマトグラフィー−質量分析法によって分析するため、その質量分析の際に試料中のアマドリ転位生成物がＡＧＥへと変化する不都合が防止され、また、夾雑物質によるピークを除去してノイズを低減することができるだけでなく、ＡＧＥの検出レベルを向上することができるため、分析対象とするＡＧＥを誤判読する危険性が低く、高感度且つ高精度なＡＧＥ分析が可能になる。
According to the sample preparation method of the present invention, there is provided a sample preparation method capable of comprehensively carrying out analysis of a free living body-derived final glycation product (AGE) with high sensitivity and high accuracy.
In addition, according to the method for analyzing a final saccharified product of the present invention, the sample prepared by the sample preparation method of the present invention is analyzed by liquid chromatography-mass spectrometry. The inconvenience that the rearrangement product is changed to AGE can be prevented, and not only can the peak due to contaminants be removed to reduce noise, but also the detection level of AGE can be improved, so that it can be analyzed. The risk of misreading AGE is low, and highly sensitive and highly accurate AGE analysis becomes possible.
本発明の試料の調製方法で調製される試料は、遊離体の最終糖化産物（ＡＧＥ）分析のための試料として有用である。本発明の試料の調製方法で調製される試料から分析される、遊離体のＡＧＥとしては、Ｎ−ε−（カルボキシメチル）リジン、Ｎ−ε−（カルボキシエチル）リジン、メチルグリオキサール−イミダゾロン、カルボキシエチルアルギニン、Ｓ−（２−スクシニル）システイン等が挙げられる。 The sample prepared by the sample preparation method of the present invention is useful as a sample for analysis of final glycation end product (AGE) of free form. The free AGE analyzed from the sample prepared by the sample preparation method of the present invention includes N-ε- (carboxymethyl) lysine, N-ε- (carboxyethyl) lysine, methylglyoxal-imidazolone, carboxy Examples thereof include ethylarginine and S- (2-succinyl) cysteine.
本発明の試料の調製方法は少なくとも、１）生体試料に対し限外濾過膜で濾過処理する工程と、２）還元処理を施す工程とを含む。本発明の試料の調製方法においては、両処理の実施順序は問わず、濾過処理及び還元処理のどちらを先に実施しても、本発明の主たる目的、即ち、分析対象物である遊離体のＡＧＥの分析中における変質防止を達成可能であるが、斯かる目的をより確実に達成する観点から、還元処理に先立って、濾過処理を実施することが好ましい。即ち、本発明においては、生体試料に対し、限外濾過膜で濾過処理した後、その膜透過画分に還元処理を施すことが好ましい。以下、本発明の試料の調製方法における各処理について説明する。 The sample preparation method of the present invention includes at least 1) a step of filtering a biological sample with an ultrafiltration membrane, and 2) a step of performing a reduction treatment. In the sample preparation method of the present invention, regardless of the order of performing both treatments, the primary purpose of the present invention, i.e. Although it is possible to prevent alteration during the analysis of AGE, it is preferable to perform a filtration treatment prior to the reduction treatment from the viewpoint of more reliably achieving such an object. That is, in the present invention, it is preferable to subject the biological sample to filtration through an ultrafiltration membrane, and then subject the membrane permeate fraction to reduction treatment. Hereinafter, each process in the sample preparation method of the present invention will be described.
［濾過処理］
本発明に係る濾過処理の対象物である生体試料は、健常体から採取されたものであっても、疾病罹患患者のような非健常体から採取されたものでも良い。また生体試料としては、生体から採取されたあらゆる細胞、組織、及び体液、例えば、皮膚、筋肉、骨、脂肪組織、脳神経系、心臓及び血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、リンパ、血液、全血、血清、血漿、リンパ液、唾液、尿、腹水、喀痰等、並びにそれらの培養物が挙げられる。このうち、全血、血清、血漿、尿が好ましく、血清、血漿がより好ましい。生体試料を濾過処理するに際しては、必要に応じ、生体試料に水等の適当な溶媒を添加して希釈し、その希釈液を濾過処理する。
[Filtration treatment]
The biological sample that is an object of the filtration treatment according to the present invention may be collected from a healthy body or may be collected from a non-healthy body such as a diseased patient. The biological sample includes all cells, tissues, and body fluids collected from the living body, for example, circulatory system such as skin, muscle, bone, adipose tissue, cranial nervous system, heart and blood vessels, lung, liver, spleen, pancreas, Examples include kidney, digestive system, thymus, lymph, blood, whole blood, serum, plasma, lymph, saliva, urine, ascites, sputum, etc., and cultures thereof. Among these, whole blood, serum, plasma, and urine are preferable, and serum and plasma are more preferable. When filtering the biological sample, if necessary, the biological sample is diluted by adding an appropriate solvent such as water, and the diluted solution is filtered.
本発明に係る濾過処理の主たる目的は、生体試料を、分析対象物である遊離体のＡＧＥと、それ以外の夾雑物質とに分画することにある。遊離体のＡＧＥは相対的に低分子量、夾雑物質は相対的に高分子量であるところ、これらを含む生体試料を限外濾過膜で濾過処理すると、遊離体のＡＧＥを含む比較的低分子量の成分は、限外濾過膜を透過することができるが、蛋白質等の夾雑物質は、比較的高分子量のため限外濾過膜を透過できず、結果として遊離体のＡＧＥを含む低分子量成分を、膜透過画分として生体試料から分取することができる。限外濾過膜即ち分子量カットフィルターとしては、市販品を適宜使用することができ、特に、少量の試料でも処理することが可能になる観点から、遠心分離チューブタイプの限外濾過膜が好ましい。本発明に使用可能な市販の遠心分離チューブタイプの限外濾過膜としては、例えば、ザルトリウス・ジャパン社製の限外濾過膜（商品名「VIVASPIN 500」）が挙げられる。 The main purpose of the filtration treatment according to the present invention is to fractionate a biological sample into a free AGE as an analysis object and other contaminants. Free AGEs have relatively low molecular weights, and contaminants have relatively high molecular weights. When biological samples containing these substances are filtered through an ultrafiltration membrane, relatively low molecular weight components containing free AGEs are present. Can pass through the ultrafiltration membrane, but contaminants such as proteins cannot pass through the ultrafiltration membrane due to its relatively high molecular weight, and as a result, low molecular weight components including free AGEs are passed through the membrane. The permeated fraction can be collected from a biological sample. As the ultrafiltration membrane, that is, the molecular weight cut filter, a commercially available product can be used as appropriate. In particular, a centrifugal tube type ultrafiltration membrane is preferable from the viewpoint that even a small amount of sample can be processed. Examples of commercially available centrifugal tube type ultrafiltration membranes that can be used in the present invention include an ultrafiltration membrane (trade name “VIVASPIN 500”) manufactured by Sartorius Japan.
濾過処理で用いる限外濾過膜の分画分子量は、より高感度且つ高精度のＡＧＥ分析を可能にする観点から、通常は１００００以下、好ましくは５０００以下、さらに好ましくは３０００以下である。例えば、生体試料を分画分子量３０００の限外濾過膜で濾過処理すると、分子量３０００を超える成分（夾雑物質）が該膜を透過できず、分子量３０００以下の成分（遊離体のＡＧＥを含む分析対象物）が膜透過画分となる。限外濾過膜の材質は特に制限されず、例えばポリエーテルスルホン（ＰＥＳ）製、トリアセチルセルロース製、セルロースアセテート製、ポリアクリルニトリル製等の限外濾過膜を用いることができ、本発明の試料の調製方法の何れかの工程で使用する試薬・溶媒等に応じて適宜、選択すれば良い。また、限外濾過膜のタイプとしては、試料が少量でも濾過処理できることから、スピンカラムタイプが好ましい。そのような限外濾過膜は分画サイズ、容量ごとに各種市販されており、これらを適宜使用することができる。例えば、前記VIVASPIN 500には、分画分子量（ＭＷＣＯ）１００００、５０００及び３０００のＰＥＳ製の限外濾過膜が存在するので、本発明ではこれらを使用することができる。 The molecular weight cutoff of the ultrafiltration membrane used in the filtration treatment is usually 10,000 or less, preferably 5000 or less, and more preferably 3000 or less, from the viewpoint of enabling AGE analysis with higher sensitivity and accuracy. For example, when a biological sample is filtered through an ultrafiltration membrane with a molecular weight cutoff of 3000, components (contaminating substances) with a molecular weight of greater than 3000 cannot pass through the membrane, and components with a molecular weight of 3000 or less (analytes containing free AGEs) Product) becomes the membrane permeation fraction. The material of the ultrafiltration membrane is not particularly limited. For example, an ultrafiltration membrane made of polyethersulfone (PES), triacetylcellulose, cellulose acetate, or polyacrylonitrile can be used, and the sample of the present invention. What is necessary is just to select suitably according to the reagent, solvent, etc. which are used at any process of this preparation method. The ultrafiltration membrane type is preferably a spin column type because it can be filtered even with a small amount of sample. Various types of such ultrafiltration membranes are commercially available for each fraction size and volume, and these can be used as appropriate. For example, the VIVASPIN 500 includes PES ultrafiltration membranes having a molecular weight cut-off (MWCO) of 10,000, 5000, and 3000, and these can be used in the present invention.
［還元処理］
本発明の試料の調製方法において、生体試料に還元処理を施す理由は次の通りである。従来、ＡＧＥを含む試料の質量分析には、液体クロマトグラフから溶離する液体試料をエレクトロスプレープローブ（ＥＳＩプローブ）によりイオン化して質量分析計に導入する、エレクトロスプレーイオン化質量分析法が利用されているところ、このＥＳＩ法を利用してＡＧＥ分析を行うと、その分析中に、液体試料に含まれていたアマドリ転位生成物がＥＳＩプローブにてＡＧＥへと変化してしまい、分析結果の信頼性が低下するという問題があった。そこで、本発明においては、生体試料に還元処理を施すことにより、ＥＳＩ法による質量分析中に生体試料中のカルボニル基のアマドリ転位を防止し、結果として生体試料中で非ＡＧＥ成分がＡＧＥに変化することを防止し、高感度且つ高精度のＡＧＥ分析の実現を図っている。
[Reduction treatment]
In the sample preparation method of the present invention, the reason why the biological sample is subjected to the reduction treatment is as follows. Conventionally, electrospray ionization mass spectrometry, in which a liquid sample eluted from a liquid chromatograph is ionized by an electrospray probe (ESI probe) and introduced into a mass spectrometer, is used for mass analysis of a sample containing AGE. However, when AGE analysis is performed using this ESI method, the Amadori rearrangement product contained in the liquid sample is changed to AGE by the ESI probe during the analysis, and the reliability of the analysis results is improved. There was a problem of lowering. Therefore, in the present invention, the biological sample is subjected to reduction treatment to prevent the Amadori rearrangement of the carbonyl group in the biological sample during mass spectrometry by the ESI method, and as a result, the non-AGE component is changed to AGE in the biological sample. To achieve high-sensitivity and high-accuracy AGE analysis.
本発明に係る還元処理としては、還元処理剤としてヒドリド還元剤を用いたヒドリド還元（hydride reduction）処理が好ましい。ヒドリド還元とは、化合物の還元を求核剤としての水素供与体により行う還元反応である。ヒドリド還元剤としては公知の物を適宜使用可能であり、例えば、水酸化ホウ素ナトリウム〔NaBH4〕、シアノ水素化ホウ素ナトリウム〔NaBH3CN〕、水素化トリエチルホウ素リチウム〔LiBH(C2H5)3〕、水素化トリ（ｓｅｃ−ブチル）ホウ素リチウム〔LiBH(sec-C4H9)3〕、水素化トリ（ｓｅｃ−ブチル）ホウ素カリウム〔KBH(sec-C4H9)3〕、水素化ホウ素リチウム、水素化ホウ素亜鉛、アセトキシ水素化ホウ素ナトリウム、水素化アルミニウムリチウム〔(LAH) LiAlH4〕、水素化ビス（２−メトキシエトキシ）アルミニウムナトリウム〔NaAlH2(OC2H4OCH3)2〕等が挙げられる。これらのヒドリド還元剤の中でも特に、水素化ホウ素ナトリウムは、還元力が高く、且つカルボニル基を還元するが、エステルやアミド基は還元しないため、本発明で好ましく用いられる。 As the reduction treatment according to the present invention, a hydride reduction treatment using a hydride reducing agent as a reduction treating agent is preferable. Hydride reduction is a reduction reaction in which a compound is reduced with a hydrogen donor as a nucleophile. As the hydride reducing agent, known substances can be used as appropriate.For example, sodium borohydride [NaBH 4 ], sodium cyanoborohydride [NaBH 3 CN], lithium triethylborohydride [LiBH (C 2 H 5 ) 3 ], lithium tri (sec-butyl) borohydride [LiBH (sec-C 4 H 9 ) 3 ], potassium tri (sec-butyl) borohydride [KBH (sec-C4H 9 ) 3 ], borohydride Lithium, zinc borohydride, sodium acetoxyborohydride, lithium aluminum hydride [(LAH) LiAlH 4 ], sodium bis (2-methoxyethoxy) aluminum hydride [NaAlH 2 (OC 2 H 4 OCH 3 ) 2 ], etc. Is mentioned. Among these hydride reducing agents, sodium borohydride is preferably used in the present invention because it has a high reducing power and reduces a carbonyl group but does not reduce an ester or amide group.
本発明に係る還元処理は、例えば、生体試料に還元剤処理剤を含む溶液を添加し、必要に応じて振盪又は攪拌した後、所定時間静置することで実施できる。還元処理に使用される還元処理剤の量、反応時間、温度等の条件は、使用する生体試料や還元処理剤の種類等に応じて決定すれば良い。例えば、生体試料として全血、血清、血漿等の血液試料を用い、その膜透過画分に対してヒドリド還元処理を施す際に、ヒドリド還元剤が２ｍＭのNaBH4溶液を用いる場合、血清試料の１／１０容量程度で良い。 The reduction treatment according to the present invention can be carried out, for example, by adding a solution containing a reducing agent treatment agent to a biological sample, shaking or stirring as necessary, and then allowing to stand for a predetermined time. Conditions such as the amount of the reducing treatment agent used in the reduction treatment, the reaction time, and the temperature may be determined according to the biological sample to be used, the type of the reducing treatment agent, and the like. For example, when a blood sample such as whole blood, serum, or plasma is used as a biological sample and the membrane permeate fraction is subjected to a hydride reduction treatment, a 2 mM NaBH 4 solution containing a hydride reducing agent is used. About 1/10 capacity is sufficient.
［生体試料への内部標準物質の添加処理］
本発明の試料の調製方法においては、該方法によって調製された試料の定量分析の精度向上を図る観点から、生体試料に濃度既知の内部標準物質を添加しても良い。この場合、内部標準物質が添加された試料の質量分析は、いわゆる内部標準法によって行われることになる。内部標準物質としては、質量分析におけるクロマトグラムが分析対象のＡＧＥと同じ挙動を示し、且つＡＧＥのそれと重ならず、且つ試料に元来含まれていない物質を用いることができ、例えば、ＡＧＥの１３Ｃの安定同位体を用いることができる。内部標準物質の添加処理の実施時期は特に制限されず、濾過処理の前若しくは後のどちらでも良いし、又は、還元処理の前若しくは後のどちらでも良いが、後述する精製処理の前に生体試料に添加することが好ましい。生体試料に内部標準物質を添加する場合、本発明の試料の調製方法の好ましい一実施態様として、「生体試料に対し、限外濾過膜で濾過処理した後、その膜透過画分に還元処理を施し、さらにその還元処理が施された試料に対し、精製処理（後述する）を施す工程を含み、且つ該還元処理の前又は後において、生体試料に内部標準物質を添加する態様」が挙げられる。
[Addition of internal standard substance to biological sample]
In the sample preparation method of the present invention, an internal standard substance having a known concentration may be added to a biological sample from the viewpoint of improving the accuracy of quantitative analysis of the sample prepared by the method. In this case, mass analysis of the sample to which the internal standard substance is added is performed by a so-called internal standard method. As an internal standard substance, a substance whose chromatogram in mass spectrometry shows the same behavior as that of the AGE to be analyzed, does not overlap with that of the AGE, and is not originally contained in the sample can be used. A stable isotope of 13 C can be used. The timing of performing the internal standard substance addition treatment is not particularly limited, and may be before or after the filtration treatment, or before or after the reduction treatment, but before the purification treatment described later, the biological sample It is preferable to add to. When an internal standard substance is added to a biological sample, a preferred embodiment of the sample preparation method of the present invention is as follows: “A biological sample is filtered through an ultrafiltration membrane, and then the membrane permeate is subjected to a reduction treatment. And a step of performing a purification treatment (to be described later) on the sample subjected to the reduction treatment, and adding an internal standard substance to the biological sample before or after the reduction treatment. .
［精製処理］
前記の濾過処理、還元処理が施された生体試料（膜透過画分）は、そのままＡＧＥ分析用試料として使用することができるが、分析感度及び精度をより一層向上させる観点から、強酸性陽イオン交換樹脂により精製することが好ましい。即ち、本発明の試料の調製方法は、限外濾過膜による濾過処理、及び還元処理を施した生体試料に、さらに強酸性陽イオン交換樹脂による精製処理を施す態様を含む。
[Purification treatment]
The biological sample (membrane permeation fraction) that has been subjected to the filtration treatment and reduction treatment can be used as it is as a sample for AGE analysis. However, from the viewpoint of further improving analysis sensitivity and accuracy, a strongly acidic cation It is preferable to purify with an exchange resin. That is, the sample preparation method of the present invention includes a mode in which a biological sample that has been subjected to filtration treatment using an ultrafiltration membrane and reduction treatment is further subjected to purification treatment using a strongly acidic cation exchange resin.
生体試料（膜透過画分）の強酸性陽イオン交換樹脂による精製処理は、基本的には、常法に従って行うことができる。強酸性陽イオン交換樹脂による精製処理は、通常、強酸性陽イオン交換樹脂に試料（濾過処理及び還元処理が施された試料）を添加した後、該樹脂を洗浄し、その後、溶離液により該樹脂に吸着した物質を溶出させ、溶出液を回収することで実施される。 The purification treatment of a biological sample (membrane permeation fraction) with a strongly acidic cation exchange resin can basically be performed according to a conventional method. In the purification treatment with a strongly acidic cation exchange resin, usually, a sample (sample subjected to filtration and reduction treatment) is added to a strongly acidic cation exchange resin, and then the resin is washed, and then the eluent is used to wash the resin. It is carried out by eluting the substance adsorbed on the resin and collecting the eluate.
強酸性陽イオン交換樹脂としては、スルホン酸型強酸性陽イオン交換樹脂が好ましい。強酸性陽イオン交換樹脂は、市販品の強酸性陽イオン交換樹脂を使用することができる。例えば、ダイヤイオン（登録商標）ＵＢＫ−５５０、ダイヤイオン（登録商標）ＳＫ１Ｂ（三菱化学）、Ｏａｓｉｓ（商標）ＭＣＸ（日本ウォーターズ社）、ＳＴＲＡＴＡ（商標）Ｘ−Ｃ（Phenomenex）、アンバーライト（登録商標）ＩＲ１２０Ｂ、アンバーライト（登録商標）２００Ｃ、ダウエックス（登録商標）ＭＳＣ−１（The Dow Chemical Company）、デュオライトＣ２６（Rohm and Haas）、ＬＥＷＡＴＩＴ（登録商標）ＳＰ−１１２（LANXESS Distribution GmbH）等が好適に使用され得る。精製に使用する樹脂の量としては、例えば血液試料を用いる場合、血清又は血漿試料１ｍＬに対して５０〜３００ｍｇが好ましく、７０〜１５０ｍｇがさらに好ましい。 As the strong acid cation exchange resin, a sulfonic acid type strong acid cation exchange resin is preferable. As the strong acid cation exchange resin, a commercially available strong acid cation exchange resin can be used. For example, Diaion (registered trademark) UBK-550, Diaion (registered trademark) SK1B (Mitsubishi Chemical), Oasis (trademark) MCX (Nippon Waters), STRATA (trademark) X-C (Phenomenex), Amberlite (registered) (Trademark) IR120B, Amberlite (registered trademark) 200C, Dowex (registered trademark) MSC-1 (The Dow Chemical Company), Duolite C26 (Rohm and Haas), LEWATIT (registered trademark) SP-112 (LANXESS Distribution GmbH) Etc. can be suitably used. As the amount of resin used for purification, for example, when a blood sample is used, 50 to 300 mg is preferable, and 70 to 150 mg is more preferable with respect to 1 mL of serum or plasma sample.
強酸性陽イオン交換樹脂は、試料（前記還元処理が施された膜透過画分）を添加する前に、予め洗浄しておくことが好ましい。例えば、樹脂量の５０倍容量以上の１００％メタノール、必要に応じて樹脂量の５０倍容量以上の溶離液に用いる酸溶液で、次いで樹脂量の５０倍容量以上の純水で、試料を添加する前のカラムに通液させ、樹脂を洗浄する。 The strongly acidic cation exchange resin is preferably washed in advance before adding a sample (a membrane permeation fraction subjected to the reduction treatment). For example, add the sample with 100% methanol of 50 times the volume of the resin, as needed, with an acid solution used for the eluent of 50 times the volume of the resin, and then with pure water of 50 times the volume of the resin Pass through the column before washing to wash the resin.
試料（前記濾過処理及び還元処理が施された試料）を強酸性陽イオン交換樹脂に添加する方法は特に限定されないが、例えば、強酸性陽イオン交換樹脂を充填したカラムに、試料を含む液体を通液させれば良い。試料が乾固されている場合には、その乾固試料に液体を添加し、乾固物を溶解させておく。強酸性陽イオン交換樹脂の場合、試料を溶解させる液体は、ｐＨ５〜９の弱酸性〜弱塩基性の塩濃度の低い液体であれば良いが、ｐＨ６〜８の中性又は中性付近のｐＨを有する塩濃度の低い液体がより好ましく、特に純水が好ましい。必要に応じて、試料を溶解させた液体をさらに遠心し、上清を回収して使用しても良い。得られた試料を含む液体を、強酸性陽イオン交換樹脂を充填したカラムに滴下し、通液させる。通液の速度は、特に限定されないが、自然滴下程度の速度が好ましく、１ｍＬ／ｍｉｎ以下がさらに好ましい。カラムに添加した試料中のＡＧＥを含む目的物質は、強酸性陽イオン交換樹脂に吸着する。 The method of adding the sample (the sample subjected to the filtration treatment and the reduction treatment) to the strong acid cation exchange resin is not particularly limited. For example, a liquid containing the sample is added to a column packed with the strong acid cation exchange resin. What is necessary is just to let it pass. When the sample is dried, a liquid is added to the dried sample to dissolve the dried product. In the case of a strongly acidic cation exchange resin, the liquid in which the sample is dissolved may be a weakly acidic to weakly basic liquid having a pH of 5 to 9, but a neutral or near neutral pH of 6 to 8. A liquid having a low salt concentration is more preferable, and pure water is particularly preferable. If necessary, the liquid in which the sample is dissolved may be further centrifuged, and the supernatant may be collected and used. The liquid containing the obtained sample is dropped onto a column packed with a strongly acidic cation exchange resin and allowed to pass therethrough. Although the speed | rate of liquid flow is not specifically limited, The speed | rate about a natural dripping is preferable and 1 mL / min or less is more preferable. The target substance containing AGE in the sample added to the column is adsorbed on the strongly acidic cation exchange resin.
次いで、目的物質が吸着した樹脂を洗浄する。洗浄は、ギ酸、例えば０．０５〜０．２Ｎ塩酸溶液、又は１．５〜２．５質量％ギ酸溶液、又はこのギ酸の終濃度となる、ギ酸とメタノールとの等量混合溶液を添加し、カラムを通過させれば良い。洗浄によりカラム中の夾雑物が除去されるので、その後の溶出処理により、目的物質を選択的に回収することが可能となる。 Next, the resin on which the target substance is adsorbed is washed. Washing is performed by adding formic acid, for example, 0.05 to 0.2N hydrochloric acid solution, or 1.5 to 2.5% by mass formic acid solution, or a mixed solution of equal amounts of formic acid and methanol to give a final concentration of this formic acid. And pass through the column. Since the contaminants in the column are removed by the washing, the target substance can be selectively recovered by the subsequent elution treatment.
溶出処理は、非酸性条件下で行うことが望ましい。例えば、洗浄処理後の樹脂に、揮発性で中性〜塩基性、好ましくはｐＨ７以上１３以下の溶離液を添加し、樹脂に吸着した目的物質を溶出させる。好ましい溶離液としては、純水、アンモニア溶液、及びこれらとメタノールの混合溶液などを挙げることができ、より好ましくは５〜１０質量％アンモニア含有溶液が挙げられる。溶離液の量や濃度は、試料や樹脂の種類によって最適化すれば良いが、樹脂に吸着した目的物質が回収される量及び濃度であれば良い。一般的には、樹脂体積の２０〜５００倍量使用すれば良い。溶離液は、カラムに自然滴下し、通液させれば良い。 The elution treatment is desirably performed under non-acidic conditions. For example, a volatile, neutral to basic, preferably pH 7 or more and 13 or less eluent is added to the washed resin to elute the target substance adsorbed on the resin. Preferable eluents include pure water, ammonia solution, and a mixed solution of these with methanol, and more preferably 5 to 10% by mass ammonia-containing solution. The amount and concentration of the eluent may be optimized depending on the type of the sample and the resin, but may be any amount and concentration at which the target substance adsorbed on the resin is recovered. Generally, it may be used in an amount 20 to 500 times the resin volume. The eluent may be dropped spontaneously on the column and allowed to pass through.
溶出液は、全画分をＡＧＥ分析用試料として使用しても良いが、目的物質の含有量の高い画分を選択的に回収してＡＧＥ分析用試料として使用することが好ましい。目的物質の含有量の高い画分は、標準溶液を用いてカラム精製を行い、経時的に分取した溶出液の各画分について目的物質の含有量を調べることによって、予め決定しておくことができる。 The eluate may use the entire fraction as a sample for AGE analysis, but it is preferable to selectively collect a fraction having a high content of the target substance and use it as a sample for AGE analysis. The fraction with a high content of the target substance should be determined in advance by performing column purification using a standard solution and examining the content of the target substance for each fraction of the eluate collected over time. Can do.
イオン交換樹脂に液体を通過させる場合、該樹脂を充填したカラムの上から液体を滴下して自然に落下させることで樹脂に液体を通過させても良いが、カラムをバキュームマニホールド等にセットし、減圧することで、効率良く液体をカラム内に導入することができる。 When passing the liquid through the ion exchange resin, the liquid may be dropped from the top of the column filled with the resin and dropped naturally, but the column is set in a vacuum manifold or the like, By reducing the pressure, the liquid can be efficiently introduced into the column.
溶離液により強酸性陽イオン交換樹脂から溶出された溶出液は、好ましくはさらなる精製処理に供される。例えば、前記手順にて得られた溶出液を、乾固処理し、次いで適切な溶媒に溶解させた後、夾雑物質等の所望しない混入物を除去する目的で、濾過処理（以下、この混入物除去目的のカラム溶出液の濾過処理を、前記の本発明に係る濾過処理と区別するために、「予備的濾過処理」ともいう）を施しても良い。予備的濾過処理としては、例えば、遠心や減圧処理による精密濾過、又は限外濾過を行うことができる。精密濾過には、エキクロディスク１３ＣＲ（孔径０．２μｍ、日本ポール社）、ミニザルトＲＣ４（孔径０．２μｍ、ザルトリウス社）、マイレクスＬＧ（孔径０．２μｍ、メルクミリポア社）等のフィルターを、限外濾過には、ナノセップＵＦ（分画分子量３Ｋ〜３００Ｋ、日本ポール社）、ビバスピン５００（分画分子量３Ｋ〜１０００Ｋ、ザルトリウス社）等のフィルターを用いることができる。使用するフィルターは、乾固試料を溶解した溶媒に対して溶媒耐性があれば特に限定されない。 The eluate eluted from the strongly acidic cation exchange resin by the eluent is preferably subjected to further purification treatment. For example, the eluate obtained in the above procedure is dried and then dissolved in a suitable solvent, and then filtered for the purpose of removing unwanted contaminants such as contaminants (hereinafter referred to as this contaminant). In order to distinguish the filtration treatment of the column eluate for the purpose of removal from the filtration treatment according to the present invention, it may be referred to as “preliminary filtration treatment”). As the preliminary filtration treatment, for example, microfiltration by centrifugation or reduced pressure treatment, or ultrafiltration can be performed. For microfiltration, filters such as Excrodisc 13CR (pore size 0.2 μm, Nippon Pall), Minisalto RC4 (pore size 0.2 μm, Sartorius), Milex LG (pore size 0.2 μm, Merck Millipore) are limited. For external filtration, a filter such as Nanosep UF (fractionated molecular weight 3K to 300K, Nihon Pall), Vivaspin 500 (fractionated molecular weight 3K to 1000K, Sartorius) can be used. The filter to be used is not particularly limited as long as it has solvent resistance to the solvent in which the dried sample is dissolved.
［ＡＧＥ分析］
前記手順により精製された試料は、ＡＧＥ分析に適切な形態へと調製され、ＡＧＥ分析に供される。ＡＧＥ分析用試料は、ＡＧＥ分析の方法や使用する機器に応じて適宜調製され得るため、その形態は特に限定されない。
[AGE analysis]
The sample purified by the above procedure is prepared into a form suitable for AGE analysis and subjected to AGE analysis. Since the sample for AGE analysis can be appropriately prepared according to the AGE analysis method and the equipment to be used, its form is not particularly limited.
ＡＧＥ分析の方法としては、ＡＧＥが測定可能な方法であれば特に限定されないが、液体クロマトグラフィーと質量分析とを組み合わせた分析方法が好ましく、例えば、液体クロマトグラフィー−質量分析（例えば、ＬＣ−ＭＳ法、ＬＣ−ＭＳ／ＭＳ、ＬＣ−ＭＳ／ＭＳ／ＭＳ等）法が挙げられる。検出感度をより向上させるためには、ＬＣ−ＭＳ／ＭＳ法や、ＬＣ−ＭＳ／ＭＳ／ＭＳ法などの液体クロマトグラフィー−タンデム型質量分析法がより好ましい。 The AGE analysis method is not particularly limited as long as AGE can be measured, but an analysis method combining liquid chromatography and mass spectrometry is preferable. For example, liquid chromatography-mass spectrometry (for example, LC-MS Method, LC-MS / MS, LC-MS / MS / MS, etc.) method. In order to further improve the detection sensitivity, liquid chromatography-tandem mass spectrometry such as LC-MS / MS method or LC-MS / MS / MS method is more preferable.
特に、液体クロマトグラフィー−質量分析法の一種であるエレクトロスプレーイオン化質量分析法、即ち、液体クロマトグラフから溶離する液体試料をエレクトロスプレープローブ（ＥＳＩプローブ）によりイオン化して質量分析計に導入する質量分析法を利用してＡＧＥ分析を行う方法では従来、前述したように、その分析中に液体試料中のアマドリ転位生成物がＥＳＩプローブにてＡＧＥへと変化することが懸念されたが、本発明の試料の調製方法によって調製されたＡＧＥ分析用の液体試料を用いた場合には、斯かる懸念が払拭される。従って、本発明の試料の調製方法によれば、ＡＧＥ分析の方法として、エレクトロスプレーイオン化質量分析法を積極的に利用することができる。 In particular, electrospray ionization mass spectrometry, which is a kind of liquid chromatography-mass spectrometry, that is, mass spectrometry in which a liquid sample eluted from a liquid chromatograph is ionized by an electrospray probe (ESI probe) and introduced into a mass spectrometer. Conventionally, in the method of performing AGE analysis using the method, as described above, there is a concern that the Amadori rearrangement product in the liquid sample is changed to AGE by the ESI probe during the analysis. When a liquid sample for AGE analysis prepared by the sample preparation method is used, such a concern is eliminated. Therefore, according to the sample preparation method of the present invention, electrospray ionization mass spectrometry can be actively used as a method for AGE analysis.
液体クロマトグラフィー−質量分析のための乾固試料溶解用の適切な溶媒としては、液体クロマトグラフィーの移動相の最終条件と同じ溶媒を用いることが好ましい。例えば、メタノールの水溶液やアセトニトリルの水溶液、アセトニトリルとトリフルオロ酢酸の混合水溶液、アセトニトリルとギ酸の混合水溶液等が挙げられるが、特に限定されない。より具体的には、本発明の試料の調製方法により調製された試料を乾固処理し、２０体積％アセトニトリル＋０．１質量％ギ酸水溶液に溶解させて、ＡＧＥ分析用試料とする。あるいは、本発明の試料の調製方法により調製された試料を乾固処理し、２０体積％アセトニトリル＋０．１質量％ギ酸溶液に溶解させた後、前述の孔径０．２μｍのフィルターを用いた精密濾過処理にかけ、回収した濾液に等量の２０体積％アセトニトリル＋０．１質量％ギ酸溶液を添加して１０００μＬまでメスアップし、ＡＧＥ分析用試料とする。血清又は血漿試料１００μＬから精製された試料に対して、９００μＬ程度の２０体積％アセトニトリル＋０．１質量％ギ酸溶液を添加すると良い。 As a suitable solvent for dissolving a dry sample for liquid chromatography-mass spectrometry, it is preferable to use the same solvent as the final condition of the mobile phase of liquid chromatography. For example, an aqueous solution of methanol, an aqueous solution of acetonitrile, a mixed aqueous solution of acetonitrile and trifluoroacetic acid, a mixed aqueous solution of acetonitrile and formic acid, and the like can be mentioned. More specifically, the sample prepared by the sample preparation method of the present invention is dried and dissolved in a 20% by volume acetonitrile + 0.1% by weight aqueous formic acid solution to obtain a sample for AGE analysis. Alternatively, after the sample prepared by the sample preparation method of the present invention is dried and dissolved in 20% by volume acetonitrile + 0.1% by mass formic acid solution, microfiltration using the aforementioned filter having a pore size of 0.2 μm is performed. Apply the treatment, add an equal volume of 20% by volume acetonitrile + 0.1% by weight formic acid solution to the collected filtrate, make up to 1000 μL, and use it as a sample for AGE analysis. About 900 μL of 20 volume% acetonitrile + 0.1 mass% formic acid solution may be added to a sample purified from 100 μL of serum or plasma sample.
液体クロマトグラフィー−質量分析計によりＡＧＥを測定する際の測定条件は、目的とするＡＧＥの種類や、機器の型、試料の状態等に応じて、当業者が通常の知識に基づいて適宜設定すれば良い。液体クロマトグラフィーの条件は供される試料によって異なるが、例えば、前述の２０体積％アセトニトリル＋０．１質量％ギ酸溶液に対しては、移動相にギ酸水溶液とギ酸アセトニトリル溶液でグラジエントを形成させると好ましい。質量分析計としては、二重収束磁場型質量分析計、イオントラップ型質量分析計、四重極型質量分析計などが挙げられるが、これらに限定されない。 The measurement conditions for measuring AGE with a liquid chromatography-mass spectrometer are appropriately set by those skilled in the art based on ordinary knowledge according to the type of AGE, the type of equipment, the state of the sample, etc. It ’s fine. The conditions for liquid chromatography vary depending on the sample to be provided. For example, for the aforementioned 20% by volume acetonitrile + 0.1% by weight formic acid solution, it is preferable to form a gradient in the mobile phase with an aqueous formic acid solution and an aqueous formic acid acetonitrile solution. . Examples of the mass spectrometer include, but are not limited to, a double focusing magnetic field mass spectrometer, an ion trap mass spectrometer, and a quadrupole mass spectrometer.
前記手順で測定された試料中のＡＧＥに関する測定値を、同様の手順で測定された標準溶液からの測定値と比較することによって、生体試料由来のＡＧＥを定量することができる。具体的には、所定濃度のＡＧＥを含有する標準溶液からの測定結果に基づいて、検量線を作成する。検量線の作成は、内部標準法又は外部標準法を利用して行うことができる。内部標準法を利用する場合は、前述したように、内部標準物質を用いて各測定値を校正しておくと、より精度の高い検量線が得られるため好ましい。 AGE derived from a biological sample can be quantified by comparing the measured value for AGE in the sample measured by the above procedure with the measured value from the standard solution measured by the same procedure. Specifically, a calibration curve is created based on the measurement result from a standard solution containing a predetermined concentration of AGE. A calibration curve can be created using an internal standard method or an external standard method. When using the internal standard method, as described above, it is preferable to calibrate each measured value using an internal standard substance because a calibration curve with higher accuracy can be obtained.
以下、本発明を具体的に説明するために実施例を挙げるが、本発明は実施例によって制限されるものではない。 EXAMPLES Hereinafter, examples are given to specifically describe the present invention, but the present invention is not limited to the examples.
〔実施例１：ラット由来生体試料の分析〕
下記（１）〜（４）の手順に従って、ＡＧＥ分析用試料を調製した。分析対象のＡＧＥは下記の通り。
１）Ｎ−ε−（カルボキシメチル）リジン〔以下、「ＣＭＬ」ともいう〕
２）メチルグリオキサール−イミダゾロン〔以下、「ＭＧ−Ｈ１」ともいう〕
３）Ｎ−ε−（カルボキシエチル）リジン〔以下、「ＣＥＬ」ともいう〕
４）カルボキシエチルアルギニン〔以下、「ＣＥＡ」ともいう〕
５）カルボキシメチルアルギニン〔以下、「ＣＭＡ」ともいう〕
[Example 1: Analysis of rat-derived biological sample]
A sample for AGE analysis was prepared according to the following procedures (1) to (4). The AGE to be analyzed is as follows.
1) N-ε- (carboxymethyl) lysine [hereinafter also referred to as “CML”]
2) Methylglyoxal-imidazolone [hereinafter also referred to as “MG-H1”]
3) N-ε- (carboxyethyl) lysine [hereinafter also referred to as “CEL”]
4) Carboxyethylarginine (hereinafter also referred to as “CEA”)
5) Carboxymethylarginine (hereinafter also referred to as “CMA”)
（１）生体試料の調製
−８０℃のディープフリーザで凍結保存されていた血清を室温で解凍・溶解した。この凍結保存されていた血清は、健常体のラット（Wister）及びストレプトゾトシン誘発糖尿病ラットをそれぞれ解剖し、腹大動脈より採血した血清を凍結保存したものである。２ｍＬチューブに溶解した血清を５０μＬ注入し、さらに、この血清に各内部標準物質を溶解した蒸留水５０μＬを添加し、よく撹拌して血清希釈液を得た。
尚、分析対象のＡＧＥがＳ−（２−スクシニル）システインである場合は、蒸留水に加えてさらにメチオニンを２０μｇ添加する。また、ここで、内部標準物質として安定同位体（１３Ｃ若しくは２Ｈ）で標識した各ＡＧＥ（ＣＭＬ、ＭＧ−Ｈ１、ＣＥＬ、ＣＥＡ、ＣＭＡ）、をそれぞれ１０ｐｍｏｌ、さらにリジンを５ ｎｍｏｌを添加している。リジンはタンパク質中に一定量存在するアミノ酸であるため、一定のタンパク量当たりのＡＧＥを定量するために必要となる。
(1) Preparation of biological sample Serum that had been frozen and stored in a deep freezer at -80 ° C was thawed and dissolved at room temperature. The cryopreserved serum was obtained by dissecting healthy rats (Wister) and streptozotocin-induced diabetic rats, and cryopreserving the serum collected from the abdominal aorta. 50 μL of serum dissolved in a 2 mL tube was injected, and further 50 μL of distilled water in which each internal standard was dissolved was added to this serum, and stirred well to obtain a serum dilution.
In addition, when AGE to be analyzed is S- (2-succinyl) cysteine, 20 μg of methionine is further added to distilled water. Also, here, 10 pmol of each AGE (CML, MG-H1, CEL, CEA, CMA) labeled with a stable isotope ( 13 C or 2 H) as internal standard substance, and further 5 nmol of lysine were added. ing. Since lysine is an amino acid present in a certain amount in a protein, it is necessary for quantifying AGE per a certain amount of protein.
（２）濾過処理
分画分子量３０００のＰＥＳ製限外濾過膜として前記VIVASPIN 500を用いて、前記血清希釈液を濾過処理した。より具体的には、限外濾過膜に前記血清希釈液を全量入れ、回転数１２０００ｒｐｍで３０分間遠心処理した後、該血清希釈液を約５０μＬ程度回収した。この遠心処理及び液回収を３回行い、計９０分間の遠心処理を行った。
(2) Filtration The serum dilution was filtered using the VIVASPIN 500 as a PES ultrafiltration membrane with a molecular weight cut-off of 3000. More specifically, the whole amount of the serum dilution was put in an ultrafiltration membrane, centrifuged at 12,000 rpm for 30 minutes, and about 50 μL of the serum dilution was recovered. This centrifugation and liquid recovery were performed three times, and a total of 90 minutes of centrifugation was performed.
（３）還元処理
濾過処理後の前記血清希釈液の入った２ｍＬチューブに、該血清希釈液と等量（例えば５０μＬ）のホウ酸ナトリウム緩衝液（0.2Mホウ酸、2mMDTPA、pH 9.0）を添加し、さらにヒドリド還元剤としての水酸化ホウ素ナトリウム溶液（2mM NaBH4、0.1 N NaOH）を、該緩衝液の１／１０量（例えば５μＬ）添加し、軽く撹拌し遠心した後、室温で４時間放置して還元処理を行った。還元処理後、吹付式試験管濃縮装置（EYELA社製, MG-2200、マニホールドS-040）にて４０℃で乾固し、未精製のＡＧＥ分析用乾固試料を得た。
(3) Reduction treatment To a 2 mL tube containing the serum diluted solution after filtration, an equivalent amount (for example, 50 μL) of sodium borate buffer solution (0.2 M boric acid, 2 mM DTPPA, pH 9.0) is added. Further, a sodium borohydride solution (2 mM NaBH 4 , 0.1 N NaOH) as a hydride reducing agent was added to 1/10 volume (for example, 5 μL) of the buffer, and the mixture was gently stirred and centrifuged, and then at room temperature for 4 hours. The reduction treatment was performed by leaving it to stand. After the reduction treatment, it was dried at 40 ° C. with a spray type test tube concentrator (EYELA, MG-2200, manifold S-040) to obtain an unpurified dry sample for AGE analysis.
（４）精製処理
強酸性陽イオン交換樹脂充填カラム（以下、「陽イオン交換カラム」ともいう）として前記ＳＴＲＡＴＡ（商標） Ｘ−Ｃを用いて、還元処理が施され内部標準物質が添加された前記ＡＧＥ分析用乾固試料を精製処理した。より具体的には、先ず、前記（３）の還元処理後、試料に蒸留水にて１０００μＬまでメスアップし、よく撹拌した。次いで、陽イオン交換カラムをサンプル分用意し、バキュームマニホールドにセットした。陽イオン交換カラムに１００％メタノールを１ｍＬずつ滴下し、バキュームマニホールドの減圧を開始した状態でチューブとの接続部のコックを開いて該メタノールを通過させた。同様の方法で、さらに陽イオン交換カラムに超純水を１ｍＬずつ滴下し、通過させ、陽イオン交換カラムを平衡にした。試料全量を陽イオン交換カラムへ滴下し通過させた。次いで、陽イオン交換に２％ギ酸を３ｍＬ通過させ、これを洗浄した。バキュームマニホールドにセットされた陽イオン交換カラムの下方に溶出液回収用の試験管をセットし、且つ該カラムの下端から延びる溶出液導出用チューブの下端を該試験管内に挿入した。そして、陽イオン交換カラムに７％アンモニア溶液を３ｍＬ滴下し、試験管内に溶出した液を回収した。その回収液を２ｍＬチューブに２ｍＬ分注し、その分注物を吹付式試験管濃縮装置にセットして４０℃で乾固させ、さらにその乾固物に、回収液を１ｍＬ追加してオーバーナイトで乾固し、ＡＧＥ分析用乾固試料を調製した。
(4) Purification treatment Using the STRATA ™ X-C as a strongly acidic cation exchange resin packed column (hereinafter also referred to as “cation exchange column”), a reduction treatment was applied and an internal standard substance was added. The dried sample for AGE analysis was purified. More specifically, after the reduction treatment (3) above, the sample was made up to 1000 μL with distilled water and stirred well. Next, a cation exchange column was prepared for the sample and set in a vacuum manifold. 1 mL of 100% methanol was added dropwise to the cation exchange column, and the vacuum at the vacuum manifold was started, and the cock at the connection with the tube was opened to allow the methanol to pass through. In the same manner, 1 mL of ultrapure water was further added dropwise to the cation exchange column and allowed to pass through to equilibrate the cation exchange column. The entire sample was dropped and passed through the cation exchange column. The cation exchange was then passed through 3 mL of 2% formic acid, which was washed. A test tube for eluate recovery was set below the cation exchange column set in the vacuum manifold, and the lower end of the eluate outlet tube extending from the lower end of the column was inserted into the test tube. Then, 3 mL of 7% ammonia solution was dropped on the cation exchange column, and the liquid eluted in the test tube was collected. Dispense 2 mL of the collected liquid into a 2 mL tube, set the dispensed product in a spray-type test tube concentrator and dry it at 40 ° C., and add 1 mL of the collected liquid to the dried product overnight. And dried to prepare a sample for AGE analysis.
〔比較例１：ラット由来生体試料の分析〕
還元処理を実施しなかった以外は実施例１と実質的に同様にして、ＡＧＥ分析用乾固試料を調製した。
[Comparative Example 1: Analysis of rat-derived biological sample]
A dry sample for AGE analysis was prepared in substantially the same manner as in Example 1 except that the reduction treatment was not performed.
〔実施例２：マウス由来生体試料の分析〕
ラットに代えてマウスの血清を用いた、即ち、健常体のマウス（ddY）及びストレプトゾトシン誘発糖尿病マウスの血清を用いた以外は実施例１と同様にして、ＡＧＥ分析用乾固試料を調製した。
[Example 2: Analysis of mouse-derived biological sample]
A dry sample for AGE analysis was prepared in the same manner as in Example 1 except that mouse serum was used in place of the rat, that is, serum from a healthy mouse (ddY) and streptozotocin-induced diabetic mouse was used.
〔実施例３：卵巣摘出ラット由来生体試料の分析〕
ストレプトゾトシン誘発糖尿病ラットに代えて、通常のラットから卵巣を摘出したラットの血清を用いた以外は実施例１と同様にして、ＡＧＥ分析用乾固試料を調製した。本実施例で使用した卵巣摘出ラットは、脂肪蓄積・肥満モデル動物である。
[Example 3: Analysis of biological sample derived from ovariectomized rat]
A dried sample for AGE analysis was prepared in the same manner as in Example 1 except that the serum of a rat obtained by extracting an ovary from a normal rat was used instead of the streptozotocin-induced diabetic rat. The ovariectomized rat used in this example is a fat accumulation / obesity model animal.
〔ＡＧＥ分析用液体試料の調製〕
実施例及び比較例で得られた乾固試料を、それぞれ、ボルテックス又はソニケーターを使って、濃度２０質量％のアセトニトリルと濃度０．１質量％のギ酸水溶液との混合液１００μＬに溶解させた。その溶液を、孔径０．２μｍのポアフィルター付き遠心チューブ（ウルトラフリーLG：メルクミリポア, UFC30LG 00）に全量入れ、遠心機（TOMY MC-150）を用い回転数１００００ｒｐｍで３分間遠心処理した。ポアフィルターを通過した溶液を回収し、その回収液に、前記のアセトニトリル−ギ酸混合液を９００μＬ添加し、ＡＧＥ分析用液体試料を得た。
[Preparation of liquid sample for AGE analysis]
The dried samples obtained in Examples and Comparative Examples were each dissolved in 100 μL of a mixture of acetonitrile having a concentration of 20% by mass and aqueous formic acid having a concentration of 0.1% by mass using a vortex or a sonicator. The entire amount of the solution was put in a centrifuge tube with a pore size of 0.2 μm and equipped with a pore filter (Ultra Free LG: Merck Millipore, UFC30LG 00), and centrifuged at 10000 rpm for 3 minutes using a centrifuge (TOMY MC-150). The solution that passed through the pore filter was collected, and 900 μL of the acetonitrile-formic acid mixed solution was added to the collected solution to obtain a liquid sample for AGE analysis.
〔ＡＧＥ分析用液体試料の分析〕
前記〔ＡＧＥ分析用液体試料の調製〕によって得られた各ＡＧＥ分析用液体試料を、エレクトロスプレーイオン化質量分析法を利用したＬＣ−ＭＳ／ＭＳにかけ、内部標準法によりＡＧＥ（ＣＭＬ、ＭＧ−Ｈ１、ＣＥＬ、ＣＥＡ、ＣＭＡ）の定量分析を行った（実施例３はＭＧ−Ｈ１、ＣＥＡ、ＣＭＡのみ）。その分析結果を図１〜図３に示す。ＬＣ−ＭＳ／ＭＳ測定条件は以下の通り。
[Analysis of liquid samples for AGE analysis]
Each AGE analysis liquid sample obtained in [Preparation of AGE analysis liquid sample] is subjected to LC-MS / MS using electrospray ionization mass spectrometry, and AGE (CML, MG-H1, (CEL, CEA, CMA) were quantitatively analyzed (Example 3 was only MG-H1, CEA, CMA). The analysis results are shown in FIGS. LC-MS / MS measurement conditions are as follows.
（ＬＣ−ＭＳ／ＭＳの測定条件）
・クロマトグラフィーカラム：ＳｅＱｕａｎｔ、ＺＩＣ−ＨＩＬＩＣ，１５０×２．１ｍｍ、５μｍ、２００ＡＰｅｅｋ Ｈｐｌｃ Ｃｏｌｕｍｎ
・カラム温度：４０℃
・移動相Ａ：０．１質量％ギ酸水溶液、移動相Ｂ：０．１質量％ギ酸含有アセトニトリル溶液
・グラジュエント条件：移動相Ａ１０％＋移動相Ｂ９０％
・流速：２００μＬ／ｍｉｎ
インジェクション量：１０μＬ
・分析時間：２０分
・溶出時間：ＣＭＬ（約１２分）、ＭＧ−Ｈ１（約１３分）、ＣＥＬ（約１２分）、ＣＥＡ（約１３分）、ＣＭＡ（約１３分）
・イオン化方法：Ｈ−ＥＳＩ
・インジェクション量：１０μＬ
・キャピラリー温度：３００℃
・イオン化エネルギー：約３５００Ｖ（陽性イオン化時）
・ＣＭＬの検出ピーク（ｍ／ｚ）：２０５
・ＭＧ−Ｈ１の検出ピーク（ｍ／ｚ）：２２９
・ＣＥＬの検出ピーク（ｍ／ｚ）：２１９
・ＣＥＡの検出ピーク（ｍ／ｚ）：２４７
・ＣＭＡの検出ピーク（ｍ／ｚ）：２３３
(Measurement conditions for LC-MS / MS)
Chromatography column: SeQuant, ZIC-HILIC, 150 × 2.1 mm, 5 μm, 200 APeek Hplc Column
-Column temperature: 40 ° C
-Mobile phase A: 0.1 mass% formic acid aqueous solution, mobile phase B: 0.1 mass% formic acid-containing acetonitrile solution-Gradient conditions: mobile phase A10% + mobile phase B90%
・ Flow rate: 200 μL / min
Injection volume: 10μL
Analysis time: 20 minutes Elution time: CML (about 12 minutes), MG-H1 (about 13 minutes), CEL (about 12 minutes), CEA (about 13 minutes), CMA (about 13 minutes)
・ Ionization method: H-ESI
・ Injection volume: 10μL
・ Capillary temperature: 300 ℃
・ Ionization energy: About 3500V (at the time of positive ionization)
CML detection peak (m / z): 205
MG-H1 detection peak (m / z): 229
CEL detection peak (m / z): 219
CEA detection peak (m / z): 247
CMA detection peak (m / z): 233
図１には実施例１及び比較例１の試料についての分析結果、図２には実施例２の試料についての分析結果、図３には実施例３の試料についての分析結果が示されている。図中、符号Ｎは健常体（卵巣非摘出）のラット又はマウスを示し、符号ＤＭは糖尿病のラット又はマウスを示し、符号ＯＶＸは卵巣摘出ラットを示す。 FIG. 1 shows the analysis results for the samples of Example 1 and Comparative Example 1, FIG. 2 shows the analysis results for the sample of Example 2, and FIG. 3 shows the analysis results for the sample of Example 3. . In the figure, symbol N indicates a healthy (non-ovarian) rat or mouse, symbol DM indicates a diabetic rat or mouse, and symbol OVX indicates an ovariectomized rat.
通常、糖尿病の生体由来の試料は、健常な生体由来の試料に比してＡＧＥを多く含有する。従って、斯かる試料を適切に調製し分析した場合には、その分析結果に斯かる傾向が反映されることになる。
図１に示す通り、実施例１の調製方法によって得られたラット由来試料の分析結果からは斯かる傾向を明確に読み取ることができ、遊離のＡＧＥを高感度且つ高精度で分析できていることがわかる。これに対し、比較例１の調製方法、即ち還元処理を行っていない調製方法によって得られたラット由来試料の分析結果は、健常な生体と糖尿病の生体とでＡＧＥの量に明確な差異が見られない場合があった。
このことから、遊離体のＡＧＥを高感度且つ高精度で網羅的に分析するためには、本発明のように、生体試料に対し、限外濾過膜で濾過処理した後、その膜透過画分に還元処理を施すことが有効であることがわかる。そして、この本発明の試料の調製方法は、ラットのみならず、マウスや脂肪蓄積・肥満モデル動物にも有効であることが、図２及び図３から明らかである。
Usually, a sample derived from a living organism with diabetes contains more AGE than a sample derived from a healthy organism. Therefore, when such a sample is appropriately prepared and analyzed, this tendency is reflected in the analysis result.
As shown in FIG. 1, this tendency can be clearly read from the analysis result of the rat-derived sample obtained by the preparation method of Example 1, and free AGE can be analyzed with high sensitivity and high accuracy. I understand. In contrast, the analysis results of the rat-derived sample obtained by the preparation method of Comparative Example 1, that is, the preparation method not subjected to reduction treatment, show a clear difference in the amount of AGE between a healthy living body and a diabetic living body. There were cases where it was not possible.
From this, in order to comprehensively analyze the AGE of the free form with high sensitivity and high accuracy, after filtering the biological sample with an ultrafiltration membrane as in the present invention, the membrane permeation fraction It can be seen that it is effective to apply a reduction treatment to. It is clear from FIGS. 2 and 3 that this sample preparation method of the present invention is effective not only for rats but also for mice and fat accumulation / obesity model animals.
Claims (6)
生体試料に対し、限外濾過膜で濾過処理する工程、還元処理を施す工程、を含む試料の調製方法。 A method for preparing a sample for final glycation product analysis comprising:
A method for preparing a sample, comprising a step of filtering a biological sample with an ultrafiltration membrane and a step of performing a reduction treatment.
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